Catalytic antioxidants and methods of use

ABSTRACT

The invention provides small molecules that act as catalytic antioxidants and methods of use thereof. The compounds can repeatedly bind and destroy reactive oxygen species by serving as substrates for enzymes of the methionine sulfoxide reductase (Msr) class. Some embodiments of the catalytic antioxidant compounds are derived from drugs with anti-inflammatory activity due to inhibition of cyclooxygenase enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the priority of U.S. provisional patentapplication No. 60/429,269 filed on Nov. 26, 2002.

FIELD OF THE INVENTION

The invention relates to the fields of biochemistry, pharmacology, andmedicine. More particularly, the invention relates to methods andcompositions for promoting health and increasing longevity by reducingoxidative damage to cells and tissues.

BACKGROUND

Oxygen is involved in a wide range of normal metabolic reactions and isessential for the survival of all aerobic organisms, including humanbeings. Reactive oxygen species (ROS), such as superoxide, are producedin abundance as a byproduct of the incomplete reduction of oxygen thathas entered the respiratory chain. Superoxide is the precursor of otherdamaging oxygen species including hydrogen peroxide, the hypochloriteion and the hydroxyl radical. Oxidase enzymes in cells such asphagocytes and nitric oxide synthases are other sources of ROS.

While low levels of ROS are present under normal physiologicalconditions, in excess, ROS can cause oxidative damage to cells andtissues by, for example, oxidizing cellular macromolecules such asnucleic acids, lipids and proteins. Cumulative damage to cells in thismanner can result in pathology. Not surprisingly then, oxidative damagehas been implicated in a wide variety of diseases and conditionsincluding chronic obstructive lung disorders such as smoker's emphysema,reperfusion damage, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS),heart attacks, stroke, several autoimmune diseases, and aging.

Regarding the latter, oxidative damage to cellular macromolecules hasbeen postulated to accelerate the aging process and shorten lifespan.For example, the level of oxidized methionine in proteins in an animalhas been observed to increase with the age of the animal. Moreover, inDrosophila, greater resistance to ROS via over-expression of superoxidedismutase and catalase has been correlated with longer lifespan, whereasgenetic disruption of superoxide dismutase and catalase has beencorrelated with shorter lifespan.

Although cells have evolved their own enzymatic antioxidant systems(e.g., superoxide dismutase, catalase, and peroxidase) to neutralizeROS, such systems may not function at ideal levels to minimize the rateof aging and the development of disease. Accordingly, there is a clearneed for non-naturally occurring compositions and methods that reduceoxidative damage to cells. One approach to increase the antioxidantactivity in cells is to provide cells with compounds that directlyscavenge ROS, e.g., vitamins C, E, and A, glutathione, ubiquinone, uricacid, carotenoids, and the like. Such conventional antioxidantcompounds, however, lose activity after neutralizing only one or two ROSmolecules. They are thus limited by the relatively small quantities ofROS that they can destroy.

SUMMARY

The invention relates to the development of methyl sulfoxide or methylsulfide containing catalytic antioxidants that can repeatedly beoxidized by a ROS, reduced back to an unoxidized form, and oxidizedagain by a ROS. Unlike a conventional antioxidant molecule, a singlecatalytic antioxidant molecule of the invention can neutralize amultitude of different ROS molecules.

The regenerative capacity of the catalytic antioxidant molecules of theinvention is based on their ability to act as substrates for themethionine sulfoxide reductase (Msr) class of enzymes. Among the variousamino acids found in proteins, methionine (Met) is one of the mostsusceptible to oxidation. Oxidation of methionine by ROS yieldsmethionine sulfoxide [Met(O)]. The Msr enzymes, including MsrA and MsrBprominent in virtually all cells, including mammalian cells, act asrepair enzymes that catalyze the reversal of the oxidation reaction,reducing Met(O) back to methionine. In addition to reducing methionine,MsrA and several other forms of Msr enzymes known in bacteria can reducea variety of other substrates, but in all cases the core functionalgroup recognized by the enzymes is a methyl sulfoxide moiety. Byreducing methyl sulfoxide moieties back to methyl sulfide, the Msrenzymes repair damaging oxidation reactions to methionine in proteins.In addition the methionine residues in proteins, via cyclic oxidationand reduction by the Msr system, can act as scavengers of ROS. In theseways the Msr system is believed to contribute to the longevity andhealth of cells by conferring resistance to ROS (reviewed in Weissbachet al., Archiv. Biochem. Biophys. 397:172 178, 2002).

The catalytic antioxidants of the invention are small molecules that actas substrates for Msr enzymes. A scheme of interaction of the compoundsof the invention with the Msr pathway is shown diagrammatically inFIG. 1. Methyl sulfide groups on the antioxidant compounds can reactwith reactive oxygen species (ROS) such as superoxide or hydrogenperoxide to form methyl sulfoxides, for example methionine sulfoxide,which occurs in proteins and in the free form in cells. Upon trappingand destruction of the ROS by a catalytic antioxidant compound of theinvention, the methyl sulfoxide formed thereon can serve as a substratefor one or more Msr enzymes. Nucleophilic attack of the methyl sulfoxideby a cysteine residue in the Msr enzyme leads to transfer of the oxygenfrom the compound to the enzyme, reducing the compound back to itsunoxidized state (FIG. 1). The compounds, thus regenerated, areavailable for repeated reuse as antioxidants. Thus, the catalyticantioxidant compounds of the invention function not only as typical ROSscavengers, but also regenerate themselves by harnessing the catalyticaction of the Msr enzymes.

Accordingly, in one aspect, the invention features non-naturallyoccurring (or purified, naturally occurring) compounds including atleast one methyl sulfide or methyl sulfoxide moiety, the compounds beinga substrate for at least one MsrA enzyme and at least one MsrB enzyme,or a pharmaceutically acceptable salt thereof, Certain embodiments ofthe compounds are based on a backbone derived from the chemicalstructure of sulindac (1(Z)-5-fluoro-2-methyl-1[[4-(methylsulfinyl)phenyl)methylene]-1-H-indenyl-3-acetic acid).

Other embodiments of the compounds are non-naturally occurring (orpurified, naturally occurring) compounds including at least one methylsulfide or methyl sulfoxide moiety, the compounds being a substrate forat least one Msr enzyme and having a backbone not based on sulindac.Various embodiments of these compounds have a backbone based on severalknown cyclooxygenase (COX) inhibitors, including acetyl salicylic acid,mefenamic acid, ibuprofen, indomethacin, and rofecoxib (VIOXX™). Theinvention also includes compositions based on these compounds in apharmaceutically acceptable carrier.

In another aspect, the invention provides a method for reducing,preventing or reversing oxidative damage in a cell. The method includesthe steps of: (a) providing a non-naturally occurring (or purified,naturally occurring) compound including in its chemical structure atleast one methyl sulfide or methyl sulfoxide moiety, the compound beinga substrate for at least one Msr enzyme; (b) providing a cell expressingat least one Msr enzyme, the cell containing or being exposed toreactive oxygen species; and (c) contacting the cell with an amount ofthe compound sufficient to reduce, prevent, or reverse oxidative damagein the cell by the reactive oxygen species.

The cell can be within an animal subject, such as a human being. Theanimal subject can have a condition or disorder associated withoxidative damage. The disorder can involve degeneration of a nerve cell.The condition affecting the subject can be age-related.

Yet another embodiment of the invention is a method for extending thelifespan of an animal. The method involves administering to the animal atherapeutically effective amount of a non-naturally occurring (orpurified, naturally occurring) compound including at least one methylsulfide or methyl sulfoxide moiety, the compound being a substrate forat least one Msr enzyme.

As used herein, the terms “methionine moiety” and “methionine analog”include all structures encompassed by general methionine formula 1described herein, including selenomethionine derivatives.

As used herein, the term “catalytic antioxidant” refers to anon-naturally occurring (or purified, naturally occurring) antioxidantcompound that can be enzymatically regenerated after it is oxidized byan oxidizing agent (for example a ROS) such that each equivalent ofantioxidant compound can destroy more than one equivalent of theoxidizing agent.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed below. The particular embodiments discussed below areillustrative only and not intended to be limiting. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions will control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the mechanism of action of acatalytic anti-oxidant, according to an embodiment of the invention.

FIG. 2 is a schematic diagram showing the cycle of catalytic antioxidantactivity of sulindac, catalyzed by an MsrA enzyme, according to anembodiment of the invention.

FIGS. 3A and 3B is two graphs showing kinetics of sulindac sulfideproduction by MsrA, according to an embodiment of the invention.

FIGS. 4A and 4B is a schematic diagram showing chemical syntheticpathways for making methionine derivatives of sulindac (compounds 2a and3a), according to an embodiment of the invention.

FIGS. 5A and 5B is a schematic diagram showing chemical syntheticpathways for making methionine derivatives of sulindac (compounds 4a and5a), according to an embodiment of the invention.

FIGS. 6A and 6B is a schematic diagram showing chemical syntheticpathways for making catalytic antioxidants based on salicylic acid andmefenamic acid (compounds 6a and 7a, respectively), according to anembodiment of the invention.

FIGS. 7A C is a schematic diagram showing chemical synthetic pathwaysfor making catalytic antioxidants based on ibuprofen, indomethacin andVIOXX™ (compounds 8a, 9a, and 10a, respectively), according to anembodiment of the invention.

FIG. 8 shows a NMR spectrum of compound 2a of the invention.

FIG. 9 is a micrograph of a TLC plate showing the presence of reductionproducts of sulindac (S) and sulindac methionine sulfoxide (SMO)following incubation with MsrA and MsrB enzymes. Results demonstratethat S is a substrate for MsrA and that SMO is a substrate for both MsrAand MsrB.

FIG. 10 is a graph showing enhanced survival of sulindac-treated fliesexposed to oxidative stress induced by paraquat.

FIG. 11 is a graph showing enhanced survival of G93A trausgenic miceover expressing a mutant superoxide dismutase with neurodegenerativedisease treated with sulindac.

FIG. 12 is a graph showing enhanced motor performance ofsulindac-treated transgenic G93A mice.

FIG. 13 is a graph showing neuronal cell counts in sections of spinalcords of G93A mice. Neuronal cell survival is significantly higher inanimals receiving sulindac.

DETAILED DESCRIPTION

The invention encompasses compositions and methods relating to catalyticantioxidants useful in reducing or preventing oxidative damage in cells.The antioxidant compounds contain active sites that capture ROS. Theantioxidant ability of the compounds is regenerated following capture ofROS by interaction with enzymes of the Msr class that reduce methylsulfoxide moieties back to the methyl sulfide.

The below described preferred embodiments illustrate variouscompositions and methods within the invention. Nonetheless, from thedescription of these embodiments, other aspects of the invention can bemade and/or practiced based on the description provided below.

Biological Methods

Methods involving conventional chemistry, cell biology and molecularbiology techniques are described herein. Such techniques are generallyknown in the art and are described in detail in methodology treatisessuch as Classics in Total Synthesis. Targets, Strategies, Methods, K. C.Nicolaou and E. J. Sorensen, VCH, New York, 1996; and The Logic ofChemical Synthesis, E. J. Coney and Xue-Min Cheng, Wiley & Sons, NewYork, 1989. Molecular biological and cell biological methods aredescribed in treatises such as Molecular Cloning: A Laboratory Manual,3rd ed., vol. 1 3, ed. Sambrook et al., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001; and Current Protocols inMolecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates).

Catalytic Antioxidants Having Methyl Sulfide or Methyl Sulfoxide Groups

The invention provides small molecules containing at least one (e.g., 1,2, 3 or more) methyl sulfoxide or methyl sulfide group that can entercells and prevent oxidative damage by a catalytic antioxidant mechanism.The methyl sulfide group on the compounds reacts with ROS, formingmethyl sulfoxide. The methyl sulfoxide-bearing compounds, in turn, actas substrates for Msr enzymes which reduce the compounds and therebyregenerate their antioxidant properties. These compounds can beadministered to cells or animals to reduce cellular damage caused byROS.

Referring to FIG. 1, these compounds serve 1) as ROS scavengers(antioxidants) by virtue of the active groups within their structuresthat destroy or react with ROS, and 2) as catalytic antioxidants byacting as substrates for Msr enzymes that reduce the oxidized compoundsback to the unoxidized form capable of further reaction with ROS. Thecatalytic nature of the antioxidant compounds of the invention is due totheir ability to serve as substrates for Msr enzymes. The corefunctional group recognized by these enzymes is methyl sulfoxide. In thecase of N-methionine-containing peptide and protein substrates, thisfunctional group is contained within the amino acid methionine.

Any compound having a methyl sulfide or methyl sulfoxide functionalgroup that is a substrate for a Msr enzyme can be used. Sulindac, anon-steroidal anti-inflammatory drug and COX inhibitor, is one exampleof a methyl sulfoxide-containing compound that serves as a substrate forMsr enzymes. Sulindac is a pro drug, and is only active as a COXinhibitor when the methyl sulfoxide moiety on the molecule is reduced tothe sulfide. Heretofore, sulindac was not known to act as a substratefor a Msr. FIG. 2 shows the reduction of sulindac to sulindac sulfide,catalyzed by Msr. As described below, sulindac was tested as a substrateagainst six known members of the Msr family identified in bacteria (E.coli) and against Msr enzymes present in mammalian (bovine) tissues.MsrA and a membrane-associated Msr of bacteria were shown to be able toreduce sulindac to the active sulfide. In mammalian tissues, reductionof sulindac was primarily attributable to the activity of MsrA.

As further described below, sulindac administration (1) protectedDrosophila against the damage from paraquat-induced ROS production, (2)prolonged the survival of spinal cord motor neurons in mice with aneurodegenerative disease caused by oxidative damage, and (3) extendedthe lifespan of the foregoing mice.

Methionine-Based Catalytic Antioxidants

In one aspect, the invention provides catalytic antioxidant compoundshaving methionine moieties or analogs of methionine. Such compounds aresubstrates for Msr enzymes that recognize the methyl sulfoxidefunctional group in methionine (for example, MsrA and MsrB). Themethionine moiety or analog found in the methionine-containingembodiments of the compounds has the following general structure:

Groups R₁, R₂, R₃, and X in general structure 1 are defined as follows:

R₁ may be CH (of either R or S configuration).

R₂ may be a normal or branched alkyl or fluoroalkyl group having 1 to 6carbons.

R₃ may be ethyl or preferably methyl, or a fluorinated derivativethereof.

X may be either S or Se in any oxidation state.

As used herein, the terms “methionine moiety” and “methionine analog”include all structures encompassed by general formula 1, includingselenomethionine analogs of methionine. General structure 1 alsoincludes esters and salts of the carboxylic acid. Oligopeptidescontaining methionine for attachment to small molecules are alsoencompassed by the invention.

Methionine-Based Catalytic Antioxidants Derived from COX Inhibitors

Inflammation and oxidative damage are known to coexist in many diseasestates and degenerative conditions. Accordingly, particularly preferredembodiments of the methionine-containing compounds of the invention arederivatives of anti-inflammatory agents such as COX inhibitors. Specificexamples of such compounds, employing scaffolds based on several COXinhibitors, and methods for their synthesis are provided in the examplesbelow. Exemplary compounds include those derived from the followingscaffolds: sulindac; acetyl salicylic acid (ortho-acetoxybenzoic acid),mefenamic acid (2-[(2,3-Dimethylphenyl)amino]benzoic acid); ibuprofen(.alpha.-methyl-4-(2-methylpropyl)-benzeneacetic acid); indomethacin(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole-3-acetic acid); androfecoxib (4-[4-(methylsulfonyl)phenyl)-3-phenyl-2(5H)-furanone, forexample, VIOXX™, sold by Merck) and celecoxib(4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-benzenesulfon-amide,for example, CELEBREX™ sold by Pfizer.

Embodiments of the invention that are sulindac derivatives can have thefollowing general formulas 2 5: Embodiments of the invention that aresulindac derivatives have the following general formulas 2-5:

Groups R₁, R₂, R₃, R₄, R₅, R₆, and X in general formulas 2, 3, 4 and 5are defined as follows:

R₁ may be CH (of either R or S configuration).

R₂ may be a normal or branched alkyl or fluoroalkyl group having 1 to 6carbons.

R₃ may be ethyl or preferably methyl, or a fluorinated derivativethereof.

R₄ may be a hydrogen or a normal or branched alkyl group having 1 to 6carbons.

R₅ may be a CH (of either R or S configuration).

R₆ is a may be a hydrogen or a normal or branched alkyl or fluoroalkylgroup having 1 to 6 carbons.

R₇ may be a nitrogen (with substituent R₄ as defined above), a CH (ofeither R or S configuration), or a normal or branched alkyl orfluoroalkyl group having 1 to 6 carbons.

X may be either S or Se in any oxidation state.

General structures 2, 3, 4 and 5 also include esters and salts of thecarboxylic acid group. The invention also encompasses sulindacderivatives containing oligomeric methionine moieties and analogs.

Embodiments of the invention that are acetyl salicylic acid derivativescan have the following general formula:

The aromatic ring of general structure 6 may contain one or morenitrogen atoms (for example pyridine or pyrazine). The aromatic carboxylgroup in general structure 6 may be oriented ortho, meta, orpara to themethionine-based moiety. Groups R₁, R₂, R₃, R₄, R₅, and X in the generalstructure are defined as follows:

R₁ may be CH (of either R or S configuration).

R₂ may be a normal or branched alkyl or fluoroalkyl group having 1 to 6carbons.

R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

R₄ may be a hydrogen or a normal or branched alkyl group having 1 to 6carbons.

R₅ may be a nitrogen (with substituent R₄ as defined above), an oxygen,or a sulfur.

X may be either S or Se in any oxidation state.

General structure 6 also includes esters and salts of the carboxylicacid group, The invention also encompasses acetyl salicylic acidderivatives containing oligomeric methionine moieties and analogs.

Embodiments of the invention that are mefenamic acid derivatives canhave the following general formula:

Both aromatic rings of general structure 7 may contain one or morenitrogen atoms (for example pyridine or pyrazine). The aromatic carboxylgroup in general structure 7 may be oriented ortho, meta, orpara to theaniline nitrogen. Groups R₁, R₂, R₃, R₄, and X in the general structureare defined as follows:

R₁ may be CH (of either R or S configuration).

R₂ may be a normal or branched alkyl or fluoroalkyl group having 1 to 6carbons.

R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

R₄ may be a hydrogen or a normal or branched alkyl group having 1 to 6carbons.

X may be either S or Se in any oxidation state.

General structure 7 also includes esters and salts of the carboxylicacid group. Mefenamic acid derivatives containing oligomeric methionineare also envisioned.

Embodiments of the invention that are ibuprofen derivatives have thefollowing general formula:

The aromatic ring of general structure 8 may contain one or morenitrogen atoms (for example pyridine or pyrazine). The sec-butyl groupin general structure 8 may be oriented ortho, meta, or para to themethionine-based moiety. Groups R₁, R₂, R₃, R₄, R₅, and X in the generalstructure are defined as follows:

R₁ may be CH (of either R or S configuration).

R₂ may be a normal or branched alkyl or fluoroalkyl group having 1 to 6carbons.

R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

R₄ may be a hydrogen or a normal or branched alkyl group having 1 to 6carbons.

R₅ may be a CH (of either R or S configuration).

X may be either S or Se in any oxidation state.

General structure 8 also includes esters and salts of the carboxylicacid group. The invention also encompasses ibuprofen derivativescontaining oligomeric methionine moieties and analogs.

Embodiments of the invention that are indomethacin derivatives can havethe following general formula:

Groups R₁, R₂, R₃, R₄, R₅, R₆, R₇ and X in general structure 9 aredefined as follows:

R₁ may be CH (of either R or S configuration).

R₂ may be a normal or branched alkyl or fluoroalkyl group having 1 to 6carbons.

R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

R₄ may be a hydrogen or a normal or branched alkyl group having 1 to 6carbons.

R₅ may be a CH (of either R or S configuration).

R₆ is a may be a hydrogen or a normal or branched alkyl or fluoroalkylgroup consisting of 1 to 6 carbons.

R₇ may be any halogen oriented ortho, meta, or para to the carbonylgroup.

X may be either S or Se in any oxidation state.

General structure 9 also includes esters and salts of the carboxylicacid group. The invention also encompasses indomethacin derivativescontaining oligomeric methionine moieties and analogs.

Embodiments of the invention that are Vioxx® derivatives can have thefollowing general formula:

The lactone ring in general structure 10 may be oriented ortho, meta, orpara to sulfonyl group. Groups R₁, R₂, R₃, R₄, and X in generalstructure 10 are defined as follows:

R₁ may be CH (of either R or S configuration).

R₂ may be a normal or branched alkyl or fluoroalkyl group having 1 to 6carbons.

R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

R₄ may be a hydrogen or a normal or branched alkyl group having 1 to 6carbons.

X may be either S or Se in any oxidation state.

Ar may be phenyl, alkyl and halogen substituted phenyl, andheteroaromatic compounds.

General structure 10 also includes esters and salts of the carboxylicacid group. The invention also encompasses Vioxx® derivatives containingoligomeric methionine moieties and analogs.

Testing of Catalytic Antioxidant Compounds

The ability of any given molecule having a chemical structure includingat least one methyl sulfoxide- and/or methyl sulfide-containing moiety,or at least one methionine and/or methionine sulfoxide moiety to act asa catalytic antioxidant can be determined empirically.

For example, a molecule containing a methyl sulfoxide group to be tested(i.e., a test molecule) can be subjected to an enzymatic assay thatindicates if the test molecule can serve as a substrate for MsrA, MsrBor other members of the Msr family (see, for instance, the NADPH assaydescribed in Example 1, and the extraction assay described in Example 2,below). A test molecule can also be subjected to an assay that indicatesthe molecule's ability to increase resistance to oxidative stress incells in vitro (for example PC-12 cells subjected to insult with MTP+)or in an animal subject (for example, Drosophila or a mammalian model ofoxidative damage) (see, for instance, the assays described in Examples7, 8 and 9 below).

Preventing/Reversing Oxidative Damage in a Cell

The catalytic antioxidant molecules of the invention can be used toreduce, prevent or reverse oxidative damage in a cell (for example, acell in an animal). In this method, a non-naturally occurring catalyticantioxidant compound is brought into contact with the cell. Afterentering the interior of the cell, the compound, if in the reduced(sulfide) form, will be oxidized to the sulfoxide by ROS (i.e., act as aROS scavenger). Subsequent reduction catalyzed by an Msr enzyme willregenerate the original sulfide. If the test molecule contains a methylsulfoxide moiety, it will be reduced to the sulfide by the Msr systemwithin the cell and subsequently act as an antioxidant. With either thesulfide or the sulfoxide as the test molecule, the oxidation/reductioncycle will permit the compound to destroy ROS catalytically, as shown inFIG. 1.

The effectiveness of particular compounds can be assessed usingconventional in vitro and in vivo assays, for example, determining acell's, or an animal's response to a challenge with an agent thatproduces ROS. For instance, to assess a test molecule for the ability toprevent oxidative damage caused by ROS in a cell, cells can be culturedby conventional means and challenged with an agent that produces ROSwithin the cells. An exemplary cellular system for testing the effect ofROS damage in nerve cells, for example, is an assay employing PC-12cells subjected to insult with MPP+, an agent that generates superoxideand other oxygen radicals. To assess the efficacy of a test compound inan animal, Drosolphila melanogaster (fruit fly) is an excellent animalmodel. The flies can be treated with an agent that produces ROS (forexample, paraquat) and then fed with a diet containing the test moleculeand monitored for their survival, compared to control flies receivingParaquat alone. Mammalian models of oxidative damage are also well knownand include inter alia a transgenic mouse model of amyotrophic lateralsclerosis (ALS) based on a mutation in the superoxide dismutase (SOD1)gene.

Animal Subjects

Because oxidative damage to cells is a ubiquitous phenomenon, theinvention is believed to be compatible with any animal subject. Anon-exhaustive list of examples of such animals includes mammals such asmice, rats, rabbits, goats, sheep, pigs, horses, cattle, dogs, cats, andprimates such as monkeys, apes, and human beings. Those animal subjectsthat have a disease or condition that relates to oxidative damage arepreferred for use in the invention as these animals may have thesymptoms of their disease reduced or even reversed. In particular, humanpatients suffering from inflammation, chronic obstructive lung diseasessuch as emphysema, reperfusion damage after heart attack or stroke,neurodegenerative diseases (for example, Parkinson's disease,Alzheimer's disease, and ALS), autoimmune diseases such as rheumatoidarthritis, lupus, and Crohn's disease, conditions related to prematurebirth, conditions caused by exposure to ultraviolet light, andage-related conditions (as but one example, age-related degenerativeconditions of the eye including age-related macular degeneration andcataract formation) are suitable animal subjects for use in theinvention. In the experiments described herein, animals used fordemonstration of beneficial effects of protection against ROS damage bythe compounds of the invention are the fruit fly and the mouse.Nonetheless, by adapting the methods taught herein to other methodsknown in medicine or veterinary science (for example, adjusting doses ofadministered substances according to the weight of the subject animal),the compounds and compositions of the invention can be readily optimizedfor use in other animals.

Administration of Compositions

The catalytic antioxidant compositions of the invention may beadministered to animals including humans in any suitable formulation.For example, the compositions may be formulated in pharmaceuticallyacceptable carriers or diluents such as physiological saline or abuffered salt solution. Suitable carriers and diluents can be selectedon the basis ofi mode and route of administration and standardpharmaceutical practice. A description of other exemplarypharmaceutically acceptable carriers and diluents, as well aspharmaceutical formulations, can be found in Remington's PharmaceuticalSciences, a standard text in this field, and in USP/NF. Other substancesmay be added to the compositions to stabilize and/or preserve thecompositions, or enhance the activity of the Msr system. One suchenhancing substance could be nicotinamide which is part of the molecule,NADPH, that supplies the reducing power to the reaction catalyzed by themembers of the Msr family.

The compositions of the invention may be administered to animals by anyconventional technique. Such administration may be oral or parenteral(for example, by intravenous, subcutaneous, intramuscular, orintraperitoneal introduction). The compositions may also be administereddirectly to the target site by, for example, surgical delivery to aninternal or external target site, or by catheter to a site accessible bya blood vessel. Other methods of delivery, for example, liposomaldelivery or diffusion from a device impregnated with the composition,are known in the art. The compositions may be administered in a singlebolus, multiple injections, or by continuous infusion (for example,intravenously or by peritoneal dialysis). For parenteral administration,the compositions are preferably formulated in a sterilized pyrogen-freeform.

Compositions of the invention can also be administered in vitro to acell (for example, to prevent oxidative damage during ex vivo cellmanipulation, for example of organs used for organ transplantation or inin vitro assays) by simply adding the composition to the fluid in whichthe cell is contained.

Effective Doses

An effective amount is an amount which is capable of producing adesirable result in a treated animal or cell (for example, reducedoxidative damage to cells in the animal or cell). As is well known inthe medical and veterinary arts, dosage for any one animal depends onmany factors, including the particular animal's size, body surface area,age, the particular composition to be administered, time and route ofadministration, general health, and other drugs being administeredconcurrently. It is expected that an appropriate dosage for parenteralor oral administration of compositions of the invention would be in therange of about 1 μg to 100 mg/kg of body weight in humans. An effectiveamount for use with a cell in culture will also vary, but can he readilydetermined empirically (for example, by adding varying concentrations tothe cell and selecting the concentration that best produces the desiredresult). It is expected that an appropriate concentration would be inthe range of about 0.0001 100 mM. More specific dosages can bedetermined by the method described below.

Toxicity and efficacy of the compositions of the invention can bedetermined by standard pharmaceutical procedures, using cells in cultureand/or experimental animals to determine the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose that effects the desiredresult in 50% of the population). Compositions that exhibit a largeLD₅₀/ED₅₀ ratio are preferred. Although less toxic compositions aregenerally preferred, more toxic compositions may sometimes be used in invivo applications if appropriate steps are taken to minimize the toxicside effects.

Data obtained from cell culture and animal studies can be used inestimating an appropriate dose range for use in humans. A preferreddosage range is one that results in circulating concentrations of thecomposition that cause little or no toxicity. The dosage may vary withinthis range depending on the form of the composition employed and themethod of administration.

EXAMPLES

The present invention is further illustrated by the following specificexamples, which should not be construed as limiting the scope or contentof the invention in any way.

Example 1 Sulindac is a Substrate for MsrA Enzyme

The enzyme methionine sulfoxide reductase (MsrA) is known to exhibitbroad specificity for compounds that contain a methyl sulfoxide group.This example provides evidence that sulindac, a known antioxidantcontaining a methyl sulfoxide moiety, can act as a substrate for MsrA.

Materials and Methods.

Reductase assay. Reaction mixtures were prepared containing 50 mMTris-Cl pH 7.4, 15 μg of E. coli thioredoxin, 1 μg E. coli thioredoxinreductase, 100 nmoles of NADPH, 1 μmole of sulindac and 100-400 ng ofMsrA in a final volume of 500 μl. Incubations were performed at 37° C.for various times.

The amount of product (sulindac sulfide) synthesized was determined bymeasuring the oxidation of NADPH spectrophotometrically at 340 nm.Because sulindac absorbs very strongly at this wavelength, the loss ofabsorbance at 340 nm could not be measured directly. To accomplish this,the sulindac and sulindac sulfide were removed from the incubations byextraction with ethyl acetate as follows. At the end of incubation, 500μl of 0.5 M Bis-Tris-Cl pH 5.5 and 3 ml of ethyl acetate were added. Thetubes were mixed (vortexed) for 5 seconds (3 times). After separation,the organic phase was removed and another 3 ml of ethyl acetate wereadded. After mixing the organic phase was again removed. The twoextractions essentially removed all of the sulindac and sulindacsulfide, leaving the NADPH in the aqueous phase, which was measured at340 nm. The loss of absorption at 340 nm, dependent on sulindac, is ameasure of sulindac reduction. (

0.062 at 340 nm=10 nmoles of sulindac sulfide formed).

Results.

With a purified enzyme, sulindac reduction can be measured by a modifiedNADPH oxidation assay. The results of a reductase assay using MsrA fromE. coli are summarized below in Table 1.

TABLE 1 Reduction of Sulindac to Sulindac Sulfide by MsrA Sulindac MsrASulindac Thioredoxin MetS(o) Time Δ sulfide Tube # (100 ng/μl) (0.2 M)(5 μg/μl) (0.2 M) (min) OD₃₄₀ OD₃₄₀ (nmol) 1 8 μl — 3 μl — 0.657 0 2 — 5μl 3 μl — 0.660 0 3 1 μl 5 μl 3 μl — 20 0.622 0.038 5.8 4 2 μl 5 μl 3 μl— 20 0.586 0.074 11.2 5 4 μl 5 μl 3 μl — 20 0.522 0.138 21.0 6 2 μl 5 μl— — 20 0.684 7 2 μl 5 μl 3 μl — 10 0.626 0.034 5.2 8 2 μl 5 μl 3 μl — 300.531 0.129 19.4

The results show that sulindac was reduced in a time- andconcentration-dependent manner by MsrA enzyme.

Example 2 Sulindac is a Substrate for Msr Enzymes in Bacteria andMammals

This example demonstrates that sulindac is a substrate for MsrA andmembrane-bound Msr in E. coli and for MsrA and possibly other Msr inmammalian tissues.

Material and Methods.

Chemicals, enzymes and substrates. Sulindac (S), sulindac sulfide (SS)and all other chemicals and E. coli thioredoxin reductase were obtainedfrom Sigma Chemicals (St. Louis, Mo.), unless noted otherwise.Thioredoxin (from E. coli) was purchased from Promega (Madison, Wis.).N-acetyl-³H-met-R, S—(O), met-R—(O), met-S—(O) DABS-met-R—(O) andDABS-met-S—(O) were prepared as previously described (Brot N. et al.,Anal. Biochem. 122 (1982) 291-294; Lavine, F. T. J. Biol. Chem. 169(1947) 477-491; Minetti C. et al., Ital. J. Biochem. 43 (1994) 273-283).

Bacterial enzymes. Recombinant MsrA and MsrB from Escherichia coli wereobtained as described previously (Grimaud, R. et al., J. Biol. Chem. 276(2001) 48915-48920; Rahman, M. A. et al., Cellular & Molecular Biology38 (1992) 529-542). Partially purified DEAE fractions of free-S-Msr(fSMsr), free-R Msr (fRMsr) and MsrA1, and a membrane vesicle associatedMsr (mern-R,S-Msr) were prepared from an E. coli MsrA/B double mutant asdescribed (Etienne, F. et al., Biochem. & Biophys. Res. Comm. 300 (2003)378-382; Spector, D. et al., Biochem. & Biophys. Res. Comm. 302 (2003)284-289). The enzyme preparations had specific activities similar thosereported earlier.

Mammalian enzymes. Calf liver, kidney and brain extracts were preparedat 4° C. Thirty grams each of calf tissue (liver, kidney, brain) wereminced using a hand-held homogenizer in 5 volumes of buffer A containing250 mM sucrose, 10 mM Tris-Cl pH 7.4 and 1 mM EDTA. The homogenates weredounced (6 strokes) and spun at 1,500×g for 10 minutes and the pelletwas discarded. The supernatants (S-10) were spun at 10,000×g for 10minutes. The S-10 supernatants were centrifuged at 100,000×g for 12hours and the resulting pellets and supernatants (S-100) were saved. TheS100 pellets were suspended in cold buffer A and centrifuged at100,000×g for 4 hours. The washed microsomal pellets (containing all ofthe ribosomes) were suspended in 2 ml of buffer A (S-100 pellet)

To prepare mitochondria, the S10 pellets were suspended in 20 ml bufferA. The suspension was layered on top of a discontinuous Ficoll gradientmade up of an equal volume of 12% Ficoll in buffer A (lower layer) and7.5% Ficoll in buffer A (upper layer). The tubes were centrifuged at24,000×g for 24 min. The pellets were resuspended in buffer A andcentrifuged at 20,000×g for 15 min. The pellets (containingmitochondria) were suspended in 2 ml of buffer A. All fractions werestored at −80° C.

Reductase assay and quantitation of sulindac sulfide formed. With crudecellular fractions when there is a large amount of NADPH oxidation, theNADPH assay described in Example 1 above cannot be used. For use withcrude cellular fractions, an extraction assay was developed based on theability of sulindac sulfide to be extracted into benzene. The reactionmixture for the reduction of sulindac to sulindac sulfide contained in atotal volume of 30 μl: 100 mM Tris-Cl, pH 7.4; 0.6 μmolesglucose-6-phosphate; 50 ng glucose-6-phosphate dehydrogenase; 30 nmolesNADPH; 2.5 μg thioredoxin, 1 μg thioredoxin reductase, 50 nmoles Sul andvarying amount of Msr enzymes. Unless stated otherwise, incubations werefor 1 hour at 37° C. At the end of the incubation 370 μl of 25 mMTris-Cl pH 8.0, 100 μl acetonitrile and 1 ml of benzene were added toeach tube. After vortexing for 30 seconds and spinning for 1 min at roomtemperature, the benzene phase was removed and the optical density wasread at 350 nm Fifty nmoles of SS or S, when carried through theextraction procedure, gave optical density readings of 0.910 and 0.030,respectively. Under these conditions, virtually all of the SS wasextracted into the benzene, while about 2.5% of S was extracted. In someexperiments using calf tissue extracts, the standard 30 μl reactionmixture volume was tripled (90 μl) to obtain statistically significantvalues. The extraction assay was not altered except for reduction of theTris buffer volume to 310 μl.

To remove the S epimer of sulindac, the sulindac (R, S mixture) wasincubated with excess MsrA (4 μg) and DTT for 60 minutes, or until thereaction reached completion. Upon completion, any further reduction seenupon addition of an enzyme fraction in a second incubation would be dueto reduction of the R epimer of sulindac.

In some experiments the product was also identified by thin layerchromatography (TLC). After incubation, both the unreacted S and SSproduct were extracted into 1 ml ethyl acetate. The ethyl acetate phasewas removed, dried in a speed vacuum at room temperature and the residuewas suspended in 5 μl of ethyl acetate which was then loaded onto a TLCplate. The plate was developed with butanol:acetic acid:water (60:15:25)as the solvent. The compounds were visualized by their yellow color. TheRf values of S and SS were 0.80 and 0.95, respectively.

Results.

Using the extraction assay described above in Methods, it was found thatrecombinant MsrA from E. coli could reduce S to SS. FIG. 3A shows a timecourse for the reaction and FIG. 3B shows the effect of MsrAconcentration on reduction of S. The reaction was dependent on thethioredoxin reducing system. The product, SS, was independentlyidentified by TLC.

S is a substrate for mem-R, S-Msr. E. coli is known to have at least 6members of the Msr family. Referring to Table 2, these proteins differin their stereo-specificity, substrate specificity, i.e., free vs.protein-bound met(O), and location within the cell, i.e., soluble ormembrane-associated. Whereas the msrA and msrB genes have been clonedand the recombinant proteins purified, the other soluble E. coli Msrenzymes (i.e., fSMsr, fRMsr and MsrA1) have been only partiallypurified, but have been separated by conventional fractionationprocedures using DEAF cellulose chromatography (Etienne, F. et al.,Biochem. & Biophys. Res. Comm. 300 (2003) 378-382; Spector, D. et al.,Biochem. & Biophys. Res. Comm. 302 (2003) 284-289). The membraneassociated Msr, which has activity toward both the R and S forms of freeand peptide bound met(O), was present as a membrane vesicle preparation.

TABLE 2 Substrate Specificity of Methionine Sulfoxide Reductases in E.coli ENZYME SUBSTRATE TYPE  Free-R-(o)  Free-S-(o) Peptide-R-(o)Peptide-S-(o) MsrA + + MsrB (+) + fRMsr + fSMsr + MsrA1 +Membrane  + + + + Msr Brackets ( ) indicate a very weak activity.

Referring to Table 3, S was compared as a substrate for highly purifiedMsrA and MsrB from E. coli and the partially purified enzymepreparations. The results showed that MsrA and the mem-R,S-Msr are ableto reduce S to SS. Very weak activity was observed with MsrA1. S was nota substrate for MsrB, which recognizes peptide-bound met-S—(O).

TABLE 3 Activity of E. coli Msr Enzymes Using Sulindac as a Substrate.ENZYME TYPE UNITS OF ACTIVITY MsrA 11.3 MsrB 0 fRMsr 0 fSMsr 0 MsrA1<0.9 Membrane 5.1 Unit of activity is defined as nmoles of SS formed perhour. Enzyme concentrations used: 250 ng MsrA; 10 μg MsrB; 290 μg fRMsr;200 μg fSMsr; 40 μg MsrA1; 50 μg membrane fraction.

Referring now to Table 4, it is seen that the membrane bound Msr of E.coli, which likely contains more than one Msr activity, reducesprimarily the R form of sulindac. In these experiments either S, whichis a mixture of the R and S epimers, or the R epimer of sulindac (seeMethods) were used as substrates. Both exhibited similar activities.Although these results support the R form being reduced, definitiveproof may require the chemical synthesis and assay of each epimer of S.

TABLE 4 Membrane Msr of E. coli Reduces Primarily the R Epimer ofSulindac. SUBSTRATE NMOLES FORMED Sulindac (R, S) 2.75 Sulindac (R) 2.41R epimer of sulindac was obtained by incubating sulindac (R, S) withexcess MsrA as described in Methods to remove the S epimer. 35 μg ofmembrane fraction was used.

Reduction of sulindac in mammalian (bovine) tissues. Results shown inTable 5 reveal that crude homogenates (S-10 fractions, see Methods) ofcalf liver, kidney and brain are able to reduce S. Of the tissuestested, kidney has the highest specific activity and brain the lowest.

TABLE 5 Sulindac Reductase Activity in Calf Tissues. TISSUE SPECIFICACTIVITY Liver 4.39 Kidney 6.53 Brain 2.31 The preparation of thevarious S-10 fractions are described in methods. Specific activity isgiven as nmoles of product formed per hour per mg of protein.

The liver extracts were fractionated and mitochondria, S-100 and S-100pellet (microsomes) were prepared as described in Methods. As shown inTable 6, all three cellular fractions were able to reduce S to SS. Theidentity of the enzyme(s) responsible for the activity was not beendetermined, but preliminary evidence indicated that MsrA was largelyresponsible, based on the observation that the addition of excessamounts of met-S—(O) inhibited the activity in all three fractions,whereas the addition of met R—(O) had only a slight effect. Thus theresponsible enzyme had met-S—(O) activity. Because free Met(O) Msrenzymes (i.e., FS Msr and FRMsr) cannot reduce sulindac (Table 2), MsrAis most likely the enzyme responsible for this activity.

TABLE 6 Subcellular Distribution Sulindac Reductase Activity in BovineLiver. LIVER FRACTIONS SPECIFIC ACTIVITY S-10 4.39 S-100 6.20Mitochondria 2.44 Microsomes 1.31 The indicated liver fractions wereprepared as described in Methods. Specific activity is given as nmolesof product synthesized per hour per mg of protein.

Example 3 Synthesis of Sulindac Methionine Catalytic Antioxidants

As shown above, sulindac is a substrate for MsrA but not for MsrB.Sulindac contains a methyl sulfoxide moiety which is recognized by MsrAenzymes, but does not contain a N-methionine sulfoxide moiety (see FIG.2), the substrate recognized by both MsrA and MsrB enzymes (Table 2).This example describes schemes for the chemical synthesis of derivativesof sulindac that are improved as substrates for multiple Msr enzymesincluding MsrB, by modification to contain an N-substituted methionine,in which the methionine amino group is in peptide or amide linkage.

Compound 2a contains a methionine group linked through the amino groupto the acetyl moiety of sulindac. Referring to FIG. 4A, compound 2a(1(Z)-5-fluoro-2-methyl-1-[4-(methylsulfinyl)phenyl]methylene]-1H-indene-3-[1-methylthiomethylenyl-2-aminoacetyl]propanoicacid) can be prepared starting from sulindac and methionine sulfoxidemethyl esters as follows. Compound 2a was synthesized as follows. To a50 ml round bottom flask under an argon atmosphere fitted with a teflonstir bar and rubber stopper, 1.4 mmol of sulindac was dissolved in 20 mlDMF followed by the addition of 1.5 mol of methionine sulfoxide methylester. Dicyclohexylcarbodiimide (1.2 mmol), triethylamine (2.0 mmol) and4-dimethylaminopyridine (0.05 mmol) were placed in the reaction flask.After 12 hours, TLC analysis (75% ethyl acetate in hexanes) showed theformation of the product at R_(f)=0.29. The reaction mixture was thenplaced onto a 2.5 cm diameter flash column filled with approximately 6inches of silica gel and topped off with a quartz sand plug. Thefollowing elution sequence was used 5% EtOAc/Hex (250 mL), 30% EtOAc/Hex(500 mL), 50% EtOAc/Hex (250 mL) and a final elution of 85% EtOAc/Hex(250 mL). HPLC analysis of the compound (gradient elution from 5% to 95%MeCN/H₂O over 45 min) gave a peak at 22.5 min with 98% purity. ProtonNMR analysis of compound 2a is shown in FIG. 8.

Another methionine derivative of sulindac, i.e., compound 3a is given inFIG. 4B. A suitable scheme for the synthesis of compound 3a with controlof the α-carbon stereochemistry is also shown in FIG. 4B. In thisparticular synthetic method, the synthesis begins with commerciallyavailable sulindac (racemic form). The sulindac is converted to itsmethyl ester by treatment with diazomethane (CH₂N₂). The methyl ester isthen treated with a strong base to form the enolate, followed byquenching with N-bromosuccinimide (NBS) leading to the α-bromoester(Kita et al., J. Am. Chem. Soc. 123.3214, 2001). The ester group of thisintermediate is then selectively reduced to the primary alcohol usingdiisobutylaluminum hydride (DIBAL-H), according to the method ofFukuyama et al., J. Am. Chem. Soc. 116:3125, 1994, to give intermediatecompound 1-3a. Compound 1-3a epoxidizes to give intermediate compound2-3a. Treatment of compound 2-3a with methyl sulfide is expected to leadto the β-hydroxysulfide compound 3-3a (Conte et al., Tetrahedron Lett.30:4859, 1989). Using para-toluene sulfonylchloride (TsCl), the hydroxylgroup in compound 3-3a is converted to the corresponding tosylate(compound 4-3a). By an extension of the method of O'Donnell (O'Donnellet al., J. Am. Chem. Soc. 111:2353, 1989), the tosylate on compound 4-3areacts with a protected diphenylimino-glycine derivative under theinfluence of a cinchona alkaloid asymmetric phase-transfer catalyst.This reaction gives the corresponding α-imino ester (compound 5-3a),with control over the stereochemistry of the α-carbon. Subsequentaqueous hydrolysis of the imino and tert-butyl ester groups gives thedesired compound 3a.

Referring now to FIG. 5A, sulindac contains a methylene group adjacentto a carboxyl that is easily converted into enolate 1-4a. Lithiumdiisopropylamide (LDA) is a base typically used to form these types ofenolates. Intermediate 1-4a should react with bromoacetyl methioninesulfoxide (A) to form the new carbon-carbon bond found in 2-4a.Hydrolysis of this intermediate with lithium hydroxide gives thecorresponding carboxylic acid derivative (compound 4a).

FIG. 5B illustrates yet another embodiment of an N-methionine derivativeof sulindac indicated as compound 5a. In compound 5a, the sulindacstructure and the N-acetyl methionine group are tethered by a diaminechain that can be of varying length. The use of such a linker moleculeprovides the ability to generate a large variety of methioninederivatives through combinatorial synthesis methods. Compound 5a may beobtained as follows (FIG. 5B). Under the action of DCC, sulinidac iscoupled to tert-butoxycalbonyl (BOC) mono-protected diamine, followed byremoval of the BOC protecting group under acidic conditions usingtrifluoroacetic acid (TFA). This intermediate is coupled to N-acetylmethionine in the presence of DCC to give compound 5a. Compound 5a caneasily be obtained as the single enantiomer (or epimers of the sulfoxideposition). The addition of N-acetyl methionine moieties is preferred, asthese moieties are expected to act as a substrate for enzymes thatrecognize N-blocked methionine sulfoxide, (such as MsrA and MsrB). Damino acids may be preferred to minimize metabolism. A racemic mixtureof the sulfoxides (i.e., both R and S forms) is preferred if it isdesired to have the compound function as a substrate for most, if notall, known Msr family enzymes that recognize free or protein-bound formsof methionine sulfoxide (whether R or S epimers).

Example 4 Synthesis of Methionine Catalytic Antioxidants Derived FromSalicylic Acid and Mefenamic Acid

This example describes chemical synthetic schemes suitable for preparingbifunctional compounds that can serve both as catalytic antioxidants andanti-inflammatory agents (COX inhibitors).

As described above, sulindac is one example of a COX inhibitor. Thisexample describes methionine derivatives of other COX inhibitors, i.e.,acetyl salicylic acid and mefenamic acid. These bifunctional antioxidantcompounds contain the amino group of methionine in the form of an amideand preferably retain the carboxyl group found in the parent compoundsthat may be critical to their inhibitory action.

Referring to FIG. 6A, starting from the methyl ester of salicylic acid,the phenol hydroxy group is expected to react with the carbon bearingthe bromine in bromoacetylmethionine sulfoxide (BAMS) to form theoxygen-carbon bond of intermediate 1-6a. In the case of mefenamic acid,the reaction with BAMS is likely to occur at the amine nitrogen to giveintermediate 1-7a (FIG. 6B). The salicyclic and mefenamic methioninesulfoxide derivatives can be converted to the respective carboxylic acidproducts 6a and 7a using a mild hydrolysis reaction with lithiumhydroxide (LiOH).

Example 5 Synthesis of Methionine Catalytic Antioxidants Derived fromIbuprofen, Indomethacin and Rofecoxib/Vioxx®

Referring now to FIG. 7, ibuprofen (FIG. 7A), indomethacin (FIG. 7B),and rofecoxib/Vioxx® (FIG. 7C) each contain a methylene group adjacentto a carboxyl or a sulfonyl group that is easily converted into enolate,shown for intermediates 1-8a and 1-10a. Lithium diisopropylamide (LDA)is a typical base used to form enolates. Intermediates 1-8a, 1-9a, and1-10a are shown to react with bromoacetyl methionine sulfoxide to formthe new carbon-carbon bonds in intermediates 2-8a, 2-9a, and 2-10a,Hydrolysis of these intermediates with lithium hydroxide gives thecorresponding carboxylic acid derivatives (compounds 8a, 9a, and 10a).

Example 6 Sulindac Methionine Sulfoxide is a Substrate for MsrA and MsrB

As shown above, sulindac is a substrate for MsrA but not for MsrB.Referring to FIG. 4A, unmodified sulindac contains a methyl sulfoxidemoiety, but does not include within its structure a methionine sulfoxidemoiety, the required substrate for Msr B enzymes. Sulindac methioninesulfoxide (SMO), an N-acetyl methionine sulfoxide derivative of sulindacdescribed in Example 4 above includes both a methyl sulfoxide and amethionine sulfoxide (see, for instance, compound 2a in FIG. 4A). Thisexample demonstrates that SMO can serve as a substrate for both MsrA andMsrB enzymes.

Materials and Methods.

Synthesis of SMO. Sulindac methionine sulfoxide (SMO) was synthesizedaccording to the synthetic pathway described in Example 3 supra.Compound 2a was used for these experiments.

Reductase assay and thin layer chromatography (TLC). Reaction mixtureswere prepared in duplicate for assay of the reduction of sulindac (S)and sulindac methionine sulfoxide (SMO). Mixtures contained in a totalvolume of 30 μl: 2M Tris-Cl pH 7.4, 200 mM DTT, 100 nmoles of S or SMO,3 μg of MsrA enzyme, or 21 μg of MsrB enzyme. Incubation was carried outfor 2 hours at 37° C., at the end of which the duplicate samples werecombined and dried in a speed-vacuum unit at room temperature. Theresidue was suspended in 50 μl of ethanol, which was then loaded onto asilica gel TLC plate. The plate was developed with butanol: aceticacid:water (60:15:25) as the solvent. The compounds were visualized bytheir yellow color.

Results:

As discussed above, it is known that MsrA can reduce methyl sulfoxidemoieties that occur as functional groups within free and peptide-boundmethionine (i.e., Met(O)), but also within other molecules. By contrast,MsrB can only reduce Met(O), and works best with Met(O) in peptidelinkage (see Table 2). Accordingly, based on the known substratespecificity of MsrA and MsrB, several different products would bepredicted upon reaction of sulindac and SMO with MsrA and MsrB. Forexample, because the structure of sulindac (S) contains only a methylsulfoxide (as seen in FIG. 2), reduction of S by MsrA results in SS.Reduction of S by MsrB would not be expected to generate a product, dueto the absence of methionine sulfoxide in S. In contrast to unmodifiedS, SMO includes both the methyl sulfoxide group of S as well as themethyl sulfoxide included in the methionine group (see, for example,compound 2a in FIG. 4A). Accordingly, reaction of SMO with MsrA couldgenerate several possible products having one or the other, or bothmethyl sulfoxide groups reduced, i.e.: sulindac sulfide methioninesulfoxide (SSMO), sulindac methionine (SM), or sulindac sulfidemethionine (SSM). With MsrB, however, only the methionine sulfoxideshould be reduced and the expected product is SM.

FIG. 9 shows TLC results from the various incubations, i.e., MsrA+S(lane 1); MsrA+SMO (lane 2); MsrB+S (lane 3) and MsrB+SMO (lane 4). InFIG. 9, the indicated substrates and reaction products are as follows:S—sulindac; SS—sulindac sulfide; SM—sulindac methionine; SSM—sulindacsulfide methionine; SMO—sulindac methionine sulfoxide; SSMO—sulindacsulfide methionine sulfoxide. The positions where the substrates,products and standards migrate on the TLC plate are indicated by arrows.

The results of the enzyme assays demonstrate the following. Lane 1 showsthe presence of SS, indicating that sulindac is a substrate for MsrA.Lane 2 reveals formation of SSM, SM, and SSMO, demonstrating that SMO isa substrate for MsrA and that both methly sulfoxide groups can bereduced. Lane 3 shows only S, demonstrating that unmodified sulindac isnot a substrate for MsrB. By contrast, lane 4 reveals that SMO is asubstrate for MsrB, shown by the formation of SM (FIG. 9) Thus it isshown that a methionine derivative of sulindac, i.e., SMO, can act as asubstrate for both MsrA and MsrB enzymes.

Example 7 Sulindac Increases Resistance to Oxidative Stress inDrosophila

This example demonstrates that sulindac, an antioxidant containing amethyl sulfoxide moiety, can extend the lifespan of flies subjected toan agent known to kill flies via production of ROS.

Materials and Methods.

Paraquat is a cytotoxic compound known to form superoxide radicalsintracellularly. Three different concentrations of paraquat (i.e., 10mM, 5 mM and 2.5 mM) were tested. Flies (Drosophila) were raised for 3days on apple juice medium (33% apple juice, 1.7% sucrose and 2.7 mg/mlmethylparaben, a mold inhibitor, in 3.5% agar) containing variousconcentrations of sulindac or no supplement (Controls). After 3 days at25° C., flies were transferred to test vials for counting.

Results.

In the group treated with 2.5 mM paraquat, approximately 80% and 25% ofthe flies in the untreated control group, respectively, were alive after3 and 6 days of paraquat exposure. By contrast, approximately 95% and60%, respectively, of the flies treated with 2 ml-M sulindac remainedalive at the 3 day and 6 day time points (FIG. 10). Similar results wereobserved in the groups exposed to higher concentrations of paraquat. Forexample, in groups exposed to 10 mM paraquat, the respective survivalrates after 2 and 3 days were approximately 50% and 17% in the controlsand 85% and 57% in the sulindac treated groups. These resultsdemonstrate that administration of a methyl sulfoxide-containingcompound that is a substrate for MsrA can lengthen the lifespan ofparaquat-exposed flies. Earlier studies showed that over expressing MsrAenzyme in transgenic flies extended their lifespan. The present dataprovide evidence that increasing the intracellular level of a substratefor the Msr system can also provide a protective effect against damagingROS species, leading to increased longevity under conditions ofoxidative stress.

Example 8 Sulindac Promotes Cell Survival in Neuronal Cells Subjected toOxidative Damage by MPP+

This example demonstrates a protective effect of sulindac on PC-12 cellsfollowing insult with MPP+, a toxic compound that selectively destroysdopaminergic neurons in vitro, and in an in vivo animal model ofParkinson's disease.

Materials and Methods.

MPP+ neurotoxin. The neurotoxin 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) when given to both humans andprimates results in a clinical syndrome closely similar to Parkinson'sdisease. The compound is metabolized to 1-methyl-4-phenylpyridinium(MPP+) by monamine oxidase B and is subsequently selectively taken up bydopaminergic terminals and concentrated in the neuronal mitochondria inthe substantia nigra. MPP+ inhibits complex 1 of the electron transportchain and is thought to cause irreversible inactivation of the complexby generating free radicals (Hartley A., Stone J. M., Heron C, Cooper J.M., and Schapira A. H. V. J. Neurosci. 63:1987-1990, 1994) MPP+increases superoxide synthesis in vivo and in vitro. MPP+ damage isdecreased in transgenic mice overexpressing superoxide dismutase,suggesting that free radicals are involved in its neurotoxicity.

Cell culture. PC-12 cells were initially grown overnight in Dulbecco'smodified Eagle's medium containing high glucose (Gibco # 11195-065) 5%fetal calf serum and 10% horse serum in 9 cm dishes. They were thentransferred to 6 cm dishes and grown in the same medium without glucosebut using sodium pyruvate as the sole energy source (Gibco # 11966-025)These cells were pretreated with sulindac(Sigma) at concentrations of0.1, 0.2, or 0.5 mM for 48 hours, the medium containing the sulindac wasremoved and replaced with fresh medium The cells were then incubated for24 hours in medium containing MPP+ at a final concentrations of 0.2 mM.Control cells were incubated in MPP+-free medium. At the end of the 24hr period, cell viability was assayed by trypan blue exclusion.

Results.

Referring to Table 7, the results showed that 0.2 mM MPP+ was highlytoxic to PC-12 cells, causing approximately 85% of the cells to die (15%cell survival) following a 24 hour treatment with this compound.Pretreatment with sulindac prior to MPP+ insult was protective againstcell death, exhibiting a dose-response with approximately 35% cell death(65% cell survival) following pretreatment with the maximumconcentration tested, i.e., 0.5 mM. In the absence of MPP, sulindac hadno effect on the viability of the cells.

TABLE 7 Effect of Sulindac on the Viability of PC-12 Cells Treated withMPP+. Sulindac (mM) Dead cells (%) Exp 1 Dead cells (%) Exp 2 0 85 870.1 67 74 0.2 55 39 0.5 34 35

Example 9 Sulindac Extends the Lifespan of a Transgenic Mouse Model ofFamilial Amyotrophic Lateral Sclerosis (ALS)

This example provides evidence that sulindac, a methyl sulfoxidecontaining compound that acts as a substrate for MsrA enzymes cansignificantly extend lifespan, increase motor neuron cell count andimprove motor performance in a mouse model of ALS based on a mutation insuperoxide dismutase (SOD1).

Materials and Methods:

ALS is an adult onset neurodegenerative disease with an unknownetiology. ALS is most commonly sporadic with about 10% of cases beinginherited as an autosomal dominant familial form. It is now known thatabout 20% of the familial cases are associated with a mutant form ofCu/Zn SOD (Rosen, D. R., et. al., (1993) Nature 362:59-62) Although theprotein harbors a mutation (over 100 different SOD mutations having beendocumented in ALS patients), it is still enzymatically active. Oxidativedamage is one of the main hypotheses for the toxicity of the mutantprotein. The animals used in this study express a mutant form of SODsimilar to patients with the disease.

Transgenic mice expressing a mutant form of SOD similar to that found inhuman ALS patients were used for this study. Transgenic male mice with aG93A human SOD1 (G1H/+) mutation (B6SJL-TgN(SOD1-G93A)1 Gur; JacksonLaboratories, ME) were used to breed with female B6SJL mice (JacksonLaboratories, ME). The F1 generations were genotyped for the G93Amutation with polymerase chain reaction (PCR) using tail DNA, and twospecific primers from the SOD1 gene.

Sulindac administration. C93A mice were treated with sulindac at twodifferent doses, i.e., 300 PPM and 450 PPM, which was mixed into theirfood beginning on postnatal day 30. Three groups were examined (i.e.,300 PPM, 450 PPM sulindac and controls). Motor performance was assessedby Rotarod testing for each group and survival time was recorded.

Motor function testing. Mice were trained for 2-3 days to becomeacquainted with the Rotarod apparatus (Columbus instruments, Columbus,Ohio). Rotarod performances were assessed in G93A mice starting at 60days of age. The testing began with placing the mice on a rod thatrotates at 12 rpms. The time period that the mice stayed on the rodbefore falling off was recorded as a measurement of the competence oftheir motor function. Three trials were performed, and the best resultof the three trials was recorded representing the status of the motorperformance. Mice were tested twice a week until they could no longerperform the task.

Survival times. The initial sign of disease in G93A transgenic mice is aresting tremor that progresses to gait impairment, asymmetrical orsymmetrical paralysis of the hind limbs, and ultimately completeparalysis at the end stage. Mice were sacrificed when they were unableto roll over within 20 seconds after being pushed on their side. Thistime point was determined to be the time of survival, at which time micewere sacrificed.

Light microscopic immunocytochemistry. Mice were perfused transcardiallywith cold 0.1M phosphate-buffered saline (PBS) for 1 minute followed bycold 4% paraformaldehyde in PBS for 10 minutes. The spinal cords wereremoved rapidly, blocked coronally, and post-fixed in 4%paraformaldehyde in PBS for 6 hours. Blocks were cryo-protected in 30%sucrose for 24 hours and were sectioned on a cryostat at a thickness of35 micrometers. All protocols were conducted within NIH guidelines foranimal research and were approved by the Institutional Animal Care andUse Committee (IACUC).

Serial transverse sections (50 μm thick) were cut on a cryostat andcollected for Nissl staining and very fourth section was analyzed forneuronal volume and number using the optical fractionator and nucleatorprobes of the Stereo Investigator System (Microbrightfield, Colchester,Vt.). Six tissue sections of the lumbar spinal cord from each mouse wereanalyzed. All cells were counted from within the ventral horn below ahorizontal line across the gray matter through the ventral border of thecentral canal. Photomicrographs were taken on a Zeiss Axiophot IImicroscope.

Statistical analysis. Statistical analysis of survival was performedusing Kaplan-Meier test for survival measured in postnatal days,Fisher's Test for mean age of death analysis, and Scheffe test for motorperformance.

Results:

Referring to FIG. 11, G93A mice treated with 450 PPM sulindac survivedan average of 131.17±10.9 days. This was a 7% increase over controlmice, which survived an average of 123.16±11 days (P=0.083). G93A micetreated with 300 PPM sulindac also exhibited extended survival (a 10%increase) relative to the controls, with mean survival time of135.17±11.4 days (P=0.02).

The results of several statistical tests of the data shown in FIG. 11are presented in Table 8.

TABLE 8 Rank Test Chi-Square DF P-Value LogranK (Mantel-Cox) 6.744 2.0343 Breslow-Gehan-Wilcoxon 7.796 2 .0203 Tarone-Ware 7.374 2 .0250Peto-Peto-Wilcoxon 7.661 2 .0217 Harrington-Fleming (rho = .5) 7.374 2.0250

The sulindac-treated groups showed a significant improvement of motorperformance, as evaluated by Rotarod performance times (FIG. 2).Microscopic analysis of spinal cord sections revealed that thesulindac-treated mice had significantly higher counts of motor neuronsas compared with G93A controls (FIG. 13). Differences between the 300PPM and 450 PPM sulindac groups were not significant (FIG. 13 and Table9).

TABLE 9 Scheffe for Motor Performance Mean Diff. Crit. Diff. P-ValueControls, Sulindac 300 PPM −59.443 54.695 .0308 S Controls, Sulindac 450PPM −73.119 53.271 .0053 S Sulindac 300 PPM, Sulindac −13.676 56.815.8267 450 PPM

OTHER EMBODIMENTS

This description has been by way of example of how the compositions andmethods of the invention can be made and carried out. Various detailsmay be modified in arriving at the other detailed embodiments, and manyof these embodiments will come within the scope of the invention.Therefore, to apprise the public of the scope of the invention and theembodiments covered by the invention, the following claims are made.

1-2. (canceled)
 3. A compound having formula 6, or a pharmaceuticallyacceptable salt thereof;

wherein: the aromatic ring includes one or more nitrogen atoms; thearomatic carboxyl group is oriented ortho, meta, orpara to themethionine-based moiety; R₁ is CH of either t or S configuration; R₂ isa normal or branched alkyl or fluoroalkyl group having 1 to 6 carbons;R₃ is methyl or ethyl or a fluorinated derivative thereof; R₄ is ahydrogen or a normal or branched alkyl group having 1 to 6 carbons; R₅is a nitrogen with substituent R₄ as defined herein, an oxygen, or asulfur; and X is S or Se in any oxidation state.
 4. The compound ofclaim 3 having formula 6a, or a pharmaceutically acceptable saltthereof:


5. (canceled)
 6. A compound having formula 7a, or a pharmaceuticallyacceptable salt thereof:


7. (canceled)
 8. A compound having formula 8a, or a pharmaceuticallyacceptable salt thereof:

9-13. (canceled)
 14. A method for reducing, preventing or reversingoxidative damage in a cell, the method comprising the steps of: (a)providing a non-naturally occurring compound comprising at least onemethyl sulfide or methyl sulfoxide moiety, the compound being asubstrate for at least one Msr enzyme; (b) providing a cell expressingat least one Msr enzyme, said cell comprising or being exposed toreactive oxygen species; and (c) contacting the cell with an amount ofthe compound sufficient to reduce, prevent, or reverse oxidative damagein the cell by said reactive oxygen species.
 15. The method of claim 14,wherein the cell is within an animal subject.
 16. The method of claim14, wherein the animal subject has a condition or disorder associatedwith oxidative damage.
 17. The method of claim 14, wherein the disorderinvolves degeneration of a nerve cell.
 18. The method of claim 14,wherein the condition is age-related.
 19. A method for extending thelifespan of an animal comprising administering to the animal atherapeutically effective amount of a non-naturally occurring compoundaccording to any one of claims 3, 4, 6, or
 8. 20. A method of treating adisease related to oxidative damage in a cell consisting essentially ofadministering to a patient in need thereof, a compound according to anyone of claims 3, 4, 6, or 8, wherein said disease is selected from:smokers, emphysema, reperfusion damage, Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ASL), heart attacks and stroke.